Plasma membrane cholesterol modulates TCR clustering and immunological synapse formation a – d , Cholesterol quantification of naive and activated CD8 + T cells by filipin III staining ( a , b ), oxidation-based method ( c ) or biotinylation-based method ( d ) ( a , b , n = 60; c , d , n = 4). PM, plasma membrane. e , CD3 and CD8 surface levels of naive wild-type and CKO CD8 + T cells. f , TCR signalling of naive CD8 + T cells stimulated with 4 μg ml −1 anti-CD3/CD28. See Supplementary Fig. 1 for gel source data. g – i , STORM analysis of TCR clustering in naive and activated CD8 + T cells. g , Representative images. h , Ripley's K -function analysis of TCR molecules. r , radius. i , The r value at the maximal L ( r ) − r value of Ripley's K -function curves (naive, WT, n = 29, CKO, n = 22; activated, WT, n = 19, CKO, n = 17). j , k , Total internal reflection fluorescence microscopy (TIRFM) analysis of immunological synapse of CD8 + T cells on stimulatory planar lipid bilayer. j , Representative images. k , Immunological synapse area ( n = 13 cells). l – n , Parameters of TCR microcluster movements of CD8 + T cells. l , Mean square displacements (MSD). TCR microcluster movements were split into directed, confined and random movements. m , Cumulative probability distribution (CPD). n , Mean scattered plots of diffusion coefficient. TCR microclusters were from 19 WT and 20 CKO cells. o , p , Cytolytic granule polarization ( o , n = 50) and degranulation ( p , n = 3) of OT-I CTLs. Data are representative of two ( a – c , j – n , p ) or three ( f – i , o ) independent experiments, and were analysed by unpaired t -test ( c , d , p ), Mann–Whitney test ( b , i , n , o ), two-way ANOVA ( k , l ) or Kolmogorov–Smirnov test ( m ). Error bars denote s.e.m. * P